Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, China.
Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China.
Microb Cell Fact. 2022 Jun 27;21(1):128. doi: 10.1186/s12934-022-01856-8.
In previous work, we developed an E. coli extracellular secretion platform XTHHly based on the hemolysin A secretion system. It can produce bioactive peptides with simple purification procedures. However, the wider application of this platform is limited by poor secretion efficiency.
In this study, we first discovered a positive correlation between the isoelectric point (pI) value of the target protein and the secretion level of the XTHHly system. Given the extremely high secretion level of S tag, we fused it at the N-terminus and created a novel SHTXTHHly system. The SHTXTHHly system significantly increased the secretion levels of antimicrobial peptides (PEW300, LL37, and Aurein 1.2) with full bioactivities, suggesting its excellent capacity for secretory production of bioactive peptides. Furthermore, RGDS, IL-15, and alcohol dehydrogenase were successfully secreted, and their bioactivities were largely maintained in the fusion proteins, indicating the potential applications of the novel system for the rapid determination of protein bioactivities. Finally, using the SHTXTHHly system, we produced the monomeric Fc, which showed a high affinity for Fcγ Receptor I and mediated the antibody-dependent immunological effects of immune cells, demonstrating its potential applications in immunotherapies.
The SHTXTHHly system described here facilitates the secretory production of various types of proteins in E. coli. In comparison to previously reported expression systems, our work enlightens an efficient and cost-effective way to evaluate the bioactivities of target proteins or produce them.
在之前的工作中，我们基于溶血素 A 分泌系统开发了一种大肠杆菌细胞外分泌平台 XTHHly，它可以通过简单的纯化程序生产具有生物活性的肽。然而，由于分泌效率差，该平台的广泛应用受到限制。
在这项研究中，我们首先发现目标蛋白的等电点（pI）值与 XTHHly 系统的分泌水平之间存在正相关关系。鉴于 S 标签的极高分泌水平，我们将其融合在 N 端，创建了一个新的 SHTXTHHly 系统。SHTXTHHly 系统显著提高了抗菌肽（PEW300、LL37 和 Aurein 1.2）的分泌水平，且具有完整的生物活性，表明其具有出色的生物活性肽分泌生产能力。此外，RGDS、IL-15 和醇脱氢酶被成功分泌，其在融合蛋白中的生物活性得到了很大的保持，表明该新型系统在快速测定蛋白质生物活性方面具有潜在的应用。最后，我们使用 SHTXTHHly 系统生产了单体 Fc，其对 Fcγ 受体 I 具有高亲和力，并介导免疫细胞的抗体依赖的免疫效应，表明其在免疫治疗中有潜在的应用。
这里描述的 SHTXTHHly 系统促进了各种类型的蛋白质在大肠杆菌中的分泌生产。与以前报道的表达系统相比，我们的工作为评估目标蛋白的生物活性或生产它们提供了一种高效、经济有效的方法。
